Research

1. Apolipoprotein E in cardiovascular and cerebrovascular disease
Heart disease and stroke are the #1 and #3 killers in the US; related health care costs run into millions of dollars. One of the aspects we investigate is the role of apoE in age-related and life style-related onset of heart disease and stroke. While it is generally acknowledged that smoking is a direct factor in the development of heart disease, the role of second hand smoke in predisposing individuals to cardiovascular disease and stroke is grossly under estimated. In addition to examining the structural basis of the role of apoE in cholesterol transport, we are interested in investigating the effect of exposure to second hand smoke on plasma apoE levels, lipoprotein and lipid profile, and apoE function. We employ rodent models exposed to environmental tobacco smoke as a surrogate for second hand smoke in comparison with humans (passive smokers, smokers and non-smokers). In a parallel approach, we employ in vitro cell culture studies to monitor apoE secretion by hepatocytes and macrophages, and, apoE-mediated uptake under normal and oxidative stress exposed conditions. Finally, we investigate the molecular basis of the role of oxidative stress by studying the effect of specific pro-oxidants on apoE structure and function by biochemical, spectroscopic and mass spectrometric analysis using recombinant apoE. Our long term objective is to define the effect of cumulative passive smoke exposure in predisposing non-smokers, such as children and the elderly, to cardiovascular disease and stroke. My program is one of the areas of research for the Center for the Prevention of Obesity, Cardiovascular Disease and Diabetes at CHORI.

2. Apolipoprotein E/amyloid peptide interaction in Alzheimer’s disease and cerebral amyloid angiopathy
In this project, we examine the propensity of apoE4- bearing individuals to develop AD and cerebrovascular diseases such as cerebral amyloid angiopathy and stroke. AD involves deposition of endogenously generated peptides (Aβ) that aggregate to form the amyloid plaques at extra neuronal sites and in the cerebral microvasculature. The oligomeric intermediates that are generated during this process are neurotoxic, and apoE appears to have a role in aggravating the amyloidogenic process. We believe that the isoform-specific role of apoE4 as a risk factor may be attributed to a combination of factors involving poor uptake and clearance of Aβ by apoE4, combined with its enhanced ability to promote and/or stabilize the neurotoxic oligomeric Aβ. In addition, the role of apoE4 may be related to poor cholesterol delivery to the neurons. We also examine the effect of Aβ in altering the interaction of apoE with cell surface localized heparan sulfate proteoglycans and its role in developing cerebral amyloid angiopathy or cerebrovascular amyloidosis. We employ a combination of molecular biology, molecular spectroscopy, protein chemistry and cell biology approach to study the role of apoE4 in amyloid pathology. The overall objective is to elucidate the molecular basis of specific interactions between apoE isoforms, cholesterol and Aβ, which is essential in devising potential therapeutic intervention strategies.

3. Analysis of α-synuclein in Parkinson’s disease
The objective of this project is to understand the role of α-synuclein in Parkinson’s disease, a neurodegenerative disorder characterized by the presence of intra neuronal inclusions called Lewy bodies, composed predominantly of α-synuclein and lipids, and by neuronal loss. We examine neuronal cell cultures subjected to varying extent and source of oxidative stress and toxic reactive oxygen intermediates to monitor the status of α-synuclein for aggregation and potential transcriptional regulation. In addition, using recombinant human protein, we study conformational alterations that accompany the transition of lipid-free to lipid- and/or membrane-associated state of α-synuclein (which displays broad structural similarities to amphipathic apolipoproteins) from a structural viewpoint. Recently, we evaluated the role of Ca2+ on α-synuclein interaction with the membrane as a potential mechanism for triggering its aggregation. We employed a powerful fluorescence spectroscopic approach to evaluate the hydrophobicity of a given microenvironment to obtain insight into the role of the acidic tail of α-synuclein (see Figure below). Oxidative stress, a phenomenon correlated with aging, is often associated with Ca2+ dysregulation. Since aging is considered one of the main risk factors associated with Parkinson’s disease, the postulate that Ca2+ likely triggers α-synuclein aggregation has implications in the pathogenesis of synucleinopathy. Our overall goal is to understand molecular mechanisms involved in protein misfolding diseases or proteopathies involved in neurodegeneration.

Schematic representation of Ca2+-mediated interaction of α-synuclein acidic tail to membranes. α-Synuclein is believed to be unstructured in the cytosol (in aqueous buffer) (A). Upon interaction with membranes containing acidic phospholipids, it is anchored to the lipid surface via its N-terminal domain, which adopts an α-helical structure (green cylinder) (B). The C-terminal domain (red line) remains unbound, and appears to be “flapping” in the surrounding aqueous milieu. The presence of Ca2+ (black dots), a key player in events occurring at the presynaptic site, triggers membrane interaction of the C-terminal acidic tail (C). Ca2+ possibly bridges an interaction between the negatively charged residues in the acidic tail and the negatively charged head groups of the phospholipids. The acidic tail is represented as a β-strand conformation (red arrow). http://pubs3.acs.org/acs/journals/hot_article.page?in_manuscript_number=bi060939i